The microtubule and actin cytoskeletons are essential for the movement, polarization, sorting, and morphogenesis of intracellular membrane compartments. Molecular motors, scaffolding proteins, and associated filaments are recruited to membranes to power diverse trafficking events that have different force, power, kinetic, and transport requirements. Defining the mechanisms of trafficking requires understanding how different motor isoforms and families work alone and in teams, and how motors and filaments work with the scaffolding proteins and adapters that link them to membranes. Determining these mechanisms requires a detailed understanding of: the cellular organization and dynamics of the cytoskeleton and membranes; the spatial, kinetic, and mechanical relationship of different motors and filaments; the structural and biophysical properties of cytoskeletal-membrane assemblies; and the biophysical parameters that define the capabilities and mechanisms of motors and scaffolds when operating under working conditions. To this end, we assembled an extraordinarily strong scientific team with expertise in cell biology, biochemistry, structural biology, structural dynamics, and technology-development to define the role of the cytoskeleton and molecular motors in trafficking. Our team includes pioneers in the use of state-of-the-art imaging, single-molecule, and structural techniques to discover how cytoskeletal proteins function in complex cellular events. High-resolution live-cell microscopy, reconstituted cytoskeletal geometries using microfabrication and dielectrophoresis, X-ray crystallography, nanometer-resolved fluorescence tracking, single-molecule fluorescence polarization, optical trapping, and advanced biochemical techniques will be applied in highly collaborative studies to understand how motors, scaffolds, and filaments work together to power membrane dynamics. The projects and investigators are interdependent and are closely linked through research goals and common technologies, and the Aims were formulated to capitalize on the unique strengths of the team members while taking advantage of extensive synergies between the groups. We will focus on the following four Aims: (1) Investigate the Dynamics of Molecular Motors in Organelle Transport and Membrane Remodeling; (2) Investigate the Structural, Biochemical, and Cellular Properties of Cytoskeleton-Membrane Scaffolds in Organelle Morphogenesis and Motility; (3) Discover the Mechanical and Biochemical Adaptations of Membrane- Associated Motors and Scaffolds; (4) Investigate the Structural Dynamics of Myosin, Dynein and Motor Collections.